Anadenanthera colubrina regulated LPS-induced inflammation by suppressing NF-κB and p38-MAPK signaling pathways

We aimed to determine the chemical profile and unveil Anadenanthera colubrina (Vell.) Brenan standardized extract effects on inflammatory cytokines expression and key proteins from immunoregulating signaling pathways on LPS-induced THP-1 monocyte. Using the RT-PCR and Luminex Assays, we planned to show the gene expression and the levels of IL-8, IL-1β, and IL-10 inflammatory cytokines. Key proteins of NF-κB and MAPK transduction signaling pathways (NF-κB, p-38, p-NF-κB, and p-p38) were detected by Simple Western. Using HPLC-ESI-MSn (High-Performance Liquid-Chromatography) and HPLC-HRESIMS, we showed the profile of the extract that includes an opus of flavonoids, including the catechins, quercetin, kaempferol, and the proanthocyanidins. Cell viability was unaffected up to 250 µg/mL of the extract (LD50 = 978.7 µg/mL). Thereafter, the extract's impact on the cytokine became clear. Upon LPS stimuli, in the presence of the extract, gene expression of IL-1β and IL-10 were downregulated and the cytokines expression of IL-1β and IL-10 were down an upregulated respectively. The extract is involved in TLR-4-related NF-κB/MAPK pathways; it ignited phosphorylation of p38 and NF-κB, orchestrating a reduced signal intensity. Therefore, Anadenanthera colubrina's showed low cytotoxicity and profound influence as a protector against the inflammation, modulating IL-1β and IL-10 inflammatory cytokines gene expression and secretion by regulating intracellular NF-κB and p38-MAPK signaling pathways.

Pathogen-Associated Molecular Patterns (PAMPs) are molecular structures that are commonly found on various pathogens such as bacteria, viruses, fungi, and parasites.These patterns are recognized by the innate immune system as potentially harmful, triggering a series of immune responses aimed at eliminating the invading pathogens 1 .In the oral cavity, various PAMPs are found in the oral microbiome, including fungal/bacterial cell wall components such as β-Glucans, Mannans and Chitin found in fungi like Candida albicans 2,3 .Another critical PAMPs in the oral microbiome are the bacterial cell wall components such as Peptidoglycans, Lipoteichoic Acids (LTA) and Lipopolysaccharides (LPS) 1,4 .The PAMPs recognition can activate strong immune responses, as well as trigger an associated inflammatory pattern of different oral conditions, such as periodontal diseases 1,4,5 .
Recognition of these PAMPs by pattern recognition receptors (PRRs) expressed on cells of the innate immune system, such as macrophages, dendritic cells, and epithelial cells, triggers signaling pathways that lead to the activation of immune responses.Recognition of oral microbiome PAMPs ligands occurs via some of the Tolllike receptors (TLRs), NOD receptors, RIG receptors, Dectin receptor and C-lectin receptors (CLRs) whereas a signaling cascade is initiated that culminates in proinflamatory cytokine and chemokine production 1,2,6 .Thus, an associated inflammatory pattern of the condition is stablished 5,[7][8][9] .
The signaling sequence through NF-κB and MAPK pathways may also be activated once the TLRs recognize other microorganisms' PAMPs such as the LPS produced by Porphyromonas gingivalis, which is a major glycolipid within the outer membrane of gram-negative bacteria.P. gingivalis is highly associated with periodontitis pathogenesis, whereas LPS presence serves as a critical virulence factor triggering an onset inflammation pattern driven by the cytokines and inflammatory mediators release that exacerbate the immune response and local tissues damage 5,[10][11][12] .Although the host immune response is an important factor against infections, when these inflammatory axes become dysregulated, they may also be associated with collateral damage to the host, exacerbation of infection effects, and it plays an important role in carcinogenesis 13 .
The development of effective anti-inflammatory drugs becomes essential in order to mitigate the risks associated with imbalances in immune response and inflammation 14,15 .Considering the pharmacological significance of plant-derived substances, significant attention has been drawn to them, especially to discovering potential therapeutic agents for treating a wide range of disorders [16][17][18] .
In this context, Anadenanthera colubrina (Vell.)Brenan, popularly known as "angico", is widely applied in Brazilian folk medicine to treat local inflammation 19,20 .In our prior study, the A. colubrina standardized extract exhibited a significant reduction in C. albicans infection and effectively modulated the Candida-induced inflammatory response in human gingival fibroblasts 21 .However, it remains unknown whether the therapeutic effect is attributed to a specific active compound or a complex mixture of active substances within the extract-the phytocomplex.Despite the anti-inflammatory properties of A. colubrina, there is limited data on its immunomodulatory effects on transcriptome and proteome levels and molecular signaling transductions.Therefore, in the present study we aimed to investigate the phytochemical profile, the in vitro immunomodulatory effect of the standardized extract of A. colubrina bark, and the underlying cell signaling transduction pathways in LPS-stimulated monocytes.

Cytotoxicity
Using THP-1 cells, we first asked if the A. colubrina extract could treat the cells without killing them.So, we evaluated its cytotoxicity, and the experiments presented LD 50 of 978.7 µg/mL and a non-toxic profile on THP-1 cells culture, with no significant effect on cell viability in concentrations up to 250 µg/mL when compared to the vehicle and the cell control (Fig. 1).

Modulatory effect on inflammatory cytokines gene expression
Following prior studies showing the extract presents anti-inflammatory effects, we decided to perform experiments for gene expression where the results showed the A. colubrina extract at 250 µg/mL presented modulatory effects on the gene expression of inflammatory cytokines.The extract itself up-regulated the gene expression of IL-1β cytokine.Upon LPS stimuli, gene expression of IL-1β and IL-10 were downregulated by A. colubrina, while it was ineffective in modulating IL-8 gene expression (Fig. 2a,b,c).

Pro and anti-inflammatory cytokines secretion and signaling pathways regulation
The expression of pro-inflammatory (IL-8 and IL-1β) and anti-inflammatory (IL-10) cytokines after treatments with A. colubrina extract (250 µg/mL) were assessed using the THP-1 culture supernatants.The extract itself did not induce any inflammatory response.Also, it did not reduce IL-8 cytokine secretion from the LPS-induced THP-1 cells group (p < 0.05).However, in the LPS-stimulated group treated with A. colubrina, IL-1β, and IL-10 cytokines levels expression were significantly reduced and increased, respectively (p < 0.05), when compared to the LPS-induced only cells group (Fig. 3a, b, c).
Therefore, the Simple Western assay data for the regulatory signaling pathways demonstrated that the phosphorylated forms of NF-κB and p38 were detected in all groups tested, with visual differences in bands' signal intensity.In addition, the LPS-induced cells group triggered NF-κB and p-38 phosphorylated forms.A. colubrina also activated the MAPK pathway by itself and in conjunction with LPS through phosphorylation of p38.The same groups also activated the NF-κB (Figs. 4, 5).

Phytochemical profile
After the above-cited exciting results, we then performed the phytochemical profile of A. colubrina (standardized hydroethanolic extract); it was assessed by HPLC-ESI-MS n (High-Performance Liquid-Chromatography) and HRESIMS (High Resolution Electrospray Ionization Mass Spectrometry), characterized by the presence of flavonoids, predominantly heterosides of catechins, quercetin, kaempferol; and proanthocyanidins (Table 1).The base peak chromatogram is shown in Fig. 6.

Discussion
The Anadenanthera colubrina is a plant that can positively affect human health.Therefore, we studied the standardized hydroethanolic extract and found the A. colubrina, in its phytocomplex, potentially interacts with TLRs.Nonetheless, it is yet unknown if its therapeutic effect can be delivered by one specific active compound or by a complex mixture of active substances contained in the extract (the phytocomplex).For this reason, identifying bioactive compounds that could act both in modulating the virulence factors of oral microrganism and on the inflammatory response of the host against the pathogen holds promise for improved therapeutic approaches. A.  colubrina capacity to reduce C. albicans infection has been shown in our previous study.However, the approach described was employed for the first time to investigate the immunomodulatory effect of A. colubrina extract and its regulation on underlying cell signaling transduction pathways in LPS-induced THP-1 monocytes.
The extract did not affect cell viability at concentrations up to 250 µg/mL, so that it can be a therapeutic application source.Our results are in accordance with several reports that also confirmed the low toxicity of A. colubrina on macrophages [22][23][24] , keratinocytes, and tumoral cell strains 25 .
Several studies have investigated the immunomodulatory effects of medicinal plant extracts using THP-1 cells [26][27][28] supporting its application as an in vitro model to study human inflammatory diseases.So, in this study, THP-1 cells were set as the in vitro cell model to evaluate the immunomodulation effects of A. colubrina, since these cells possess regulatory proteins which initiate inflammation upon stimulation by LPS 26 .on the regulation of pro-inflammatory cytokine expression during LPS stimulation, through gene expression (transcriptional level) and secretion (proteomic level) of these inflammation mediators.
Considering that TLRs exert pro-inflammatory effects when activated, and anti-inflammatory effects when downregulated or suppressed, they are thought to play a central role in both mediating and modulating inflammatory response 29 .In this regard, we chose a panel of pro-inflammatory markers, since the interaction between TRL4 receptor and LPS induces the activation of NF-κB/MAPK signaling pathways and the release of TNF-α,    12,30,31 .Finally, we evaluated the secretion levels of IL-10 cytokine, as an antiinflammatory marker.A. colubrina was not effective in reducing the gene expression of IL-8 pro-inflammatory cytokines in the LPSinduced group.However, upon LPS stimuli, A. colubrina affected the expression of inflammatory cytokines by down-regulating IL-1β and IL-10 genes expression, which suggests the anti-inflammatory effect of the extract on the transcriptional level of regulation.In addition, our results showed that LPS stimulus effectively up-regulates the gene expression of IL-8 and IL-1β pro-inflammatory cytokines, which proves its effect on inducing transcription of genes related to inflammation responses upon interaction with TRL4 receptor 32 .
Also, we showed that A. colubrina itself did not induce any pro-inflammatory and anti-inflammatory response, which is positive considering that the extract did not significantly affect THP-1 cell viability and function.However, upon A. colubrina treatment, secretion levels of IL-8 cytokines were not affected in LPS-induced groups, which means that the extract was ineffective in decreasing this pro-inflammatory cytokine in response to the inflammatory stimuli produced by LPS.On the other hand, it is known that IL-1β is vital for the inflammatory host response against pathogens since it is involved in the recruitment of immune cells to the site of infection 33 .Therefore, the study showed a significant decrease in IL-1β secretion by A. colubrina, which may prevent additional inflammatory responses induced by the recruitment of immune cells.In addition, IL-10, a representative anti-inflammatory cytokine that plays a critical role in controlling immune responses and is reported to be involved in the inhibition of IL-1β production 34,35 , was significantly enhanced in the present study Therefore, these findings suggest that A. colubrina may regulate the immune response by modulating IL-1β and IL-10 cytokines level secretion.
The signaling pathway assay (Simple Western Immunoassay) was used to examine A. colubrina-mediatedsignal transduction and determine which pathways were affected on the regulation of pro-inflammatory cytokine gene expression, accessing the expression of key proteins involved in NF-κB/MAPK signaling pathways through the detection of phosphorylated forms of NF-κB and p38.MAPK (JNK, ERK, and p38) and NF-κB are crucial intracellular pathways leading to the inflammatory response 36 .These biological responses are mediated by their transcription factors, such as activator protein-(AP1) and NF-κB subunit Iκβα, which are phosphorylated and translocated from the cytoplasm to the nucleus, resulting in an inflammatory action through the expression of target genes, including pro-inflammatory cytokines IL-1β, IL-6, and TNF-α as well as iNOS (inducible nitric oxide synthase) and COX-2 (cyclooxygenase-2) proteins 37,38 .
In this study, we evidenced the boosted activity of phosphorylated NF-κB (NF-κB + p) and p38 (p38 + p) forms upon LPS stimulation alone in THP-1 cells, which proves the activation from NF-κB/MAPK pathways by LPS, resulting in over-production of inflammatory mediators.In addition, we observed phosphorylation from NF-κB and p38, with an attenuated signal intensity band regarding A. colubrina itself and in conjunction with LPS stimuli, especially on the NF-κB transduction factor.These findings suggest that the extract may exert molecular mechanisms involving the modulation of these signaling pathways, particularly on NF-κB protein activation.
Indeed, IL-1β is commonly induced by activating the inflammatory transcription factor NF-κB, which regulates inflammatory responses such as cell proliferation, migration, adhesion, and lymphocyte development 39 .Thus, the decrease in the release of IL-1β cytokine in Luminex analysis in our study is consistent with the downregulation of the NF-κB signaling pathway.Furthermore, considering that the extract combined with LPS was ineffective in decreasing IL-8 levels but significantly decreased IL-1β secretion.Therefore, we hypothesized that phosphorylation of p38, in conjunction with an attenuated activation of NF-κB, was responsible for the interaction between these pathways, determining this final biological response after LPS mediator stimulation 40 .
This approach may also help identify potential therapeutic targets in pathophysiological contexts of exacerbated or chronic inflammation 15,41 .Thus, we reinforce that further analysis to measure the phosphorylation levels of NF-κB and p38 proteins is necessary to verify quantitatively how the extract can regulate the expression of these key proteins related to signaling pathways that trigger the expression and production of pro-inflammatory cytokines.
Besides regulating IL-1β, IL-6, and IL-8, NF-κB/MAPK signaling cascades also monitor the expression of pro-inflammatory cytokine TNF-α 27 .Our study did not evaluate the TNF-α expression.However, recent studies that evaluated the anti-inflammatory effects of A. colubrina leaves extract 23 and protease inhibitors extracted from the same species 24 showed a significant reduction in TNF-α and nitric oxide (NO) production levels in LPS-induced macrophages.Along with these findings, our results can contribute to understanding the molecular mechanisms underlying the anti-inflammatory effects of A. colubrina.
Previously, we evaluated the phytochemical composition of the A. colubrina bark extract 25 .It was found a high total polyphenol content (53.18% gallic acid equivalents); tannins (8.77% catechin equivalents) and flavonoids (0.28% quercetin equivalents: mainly heterosides of catechin, quercetin, and kaempferol), and proanthocyanidins.In this current study, we also assessed the chemical profile of the extract by HPLC coupled to Ion Trap and TOF Mass Spectrometry (HPLC-IT-MS/HPLC-TOF-MS).Flavonoids are polyphenolic compounds commonly present in most plants with anti-inflammatory properties [42][43][44] , supporting the A. colubrina immunomodulatory property here evaluated.Additionally, flavonoids can modulate the immune response through the inhibition of molecules that play an essential role in the modulation of mediators related to inflammation response, such as regulatory enzymes and transcription factors, such as NF-κB 10,[44][45][46] .
Several studies have reported that extracts containing flavonoids, such as catechin and glycosylated derivatives of quercetin, can modulate several inflammatory and oxidative stress mediators through the negative regulation of pro-inflammatory cytokines and chemokines (TNF-α, IL-6, IL-1β, IL-8), nitric oxide (NO) and COX-2 in LPS-activated macrophages 42,43,47 .Thus, consistent with these studies, the immunomodulatory effects of A. colubrina extract observed in the present study could be related to the presence of flavonoids.
In conclusion, the present in vitro study is the first to show that A. colubrina extract has anti-inflammatory properties in LPS-induced THP-1 cells.Furthermore, these effects can be related to modulations in the secretion Vol:.( 1234567890 Technology, Danvers, MA).The samples, the blocking reagent, the primary antibodies, the HRP-conjugated secondary antibodies, and the chemiluminescent substrate were added to the plate.The WES machine and software (Compass™) provided data as virtual blots (electropherograms) in which the molecular weight and signal intensity are presented 53,54 .(n = 3 for each group, analyzed on three independent experiments).

Phytochemical profile analysis by LC-ESI-MS n and LC-HRESIMS
The phytochemical profiles of A. colubrina extract were analyzed using an HPLC (Shimadzu, Kyoto, Japan) equipped with a C18 column (Kromasil-250 mm × 4.6 mm × 5 μm) coupled to Ion-Trap (Amazon X, Bruker, Berlin, Germany) or microTOF mass spectrometers (Bruker, Berlin, Germany) with an Electrospray Ionization (ESI).First the extract was diluted in methanol (1 mg/mL) and then filtered in a 0.45 µM PVDF (Polyvinylidene Difluoride) membrane.For the chromatography method, methanol (solvent B) and ultrapure milli-Q water were used; the solution was acidified with formic acid (0.1% v/v), following gradient elution of concentration (5 to 100% of solvent B in 60 min).The injection volume was 10 µL, and the flow was set to 0.6 mL/min.Ion Trap and TOF acquisition parameters were as follows: negative ionization mode; spray voltage of 4.4 kV; offset of 500 V; sheath gas at 35 pi; drying gas N 2 ; the flow of 8 mL/min and heater temperature of 300 °C.The analysis of the compounds was based on MS/MS data available in the literature.

Statistical analysis
Data were expressed as the mean ± SEM using one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison tests to the vehicle using GraphPad Prism software (version 8.02).(p ≤ 0.05 was set as the threshold of significance).

Figure 2 .
Figure 2. Real-time quantitative information about to relative gene expression of a) IL-8, b) IL-1β, and c) IL-10 of THP-1 cells after 6 h of treatment with A. colubrina extract (250 µg/mL) and stimulation by LPS (100 ng/mL).Values are shown as the fold-change relative to the negative control group (6 h).Values shown with an asterisk (*) are statistically significant when compared to the negative control (p < 0.05).

Figure 3 .
Figure 3. Pro and anti-inflammatory cytokines expression of a) IL-8, b) IL-1β, and c) IL-10 by THP-1 cells after 6 h of treatment with A. colubrina extract (250 µg/mL).Values shown with an asterisk (*) are statistically significant when compared to the LPS-induced cells group (p < 0.05).